1. Field of the Invention
The present invention relates to a highly sensitive and stable enzyme electrode for use in the determination of alcohol content, and moreover to a method for the determination of alcohol content having high accuracy and high sensitivity.
2. Description of the Prior Art
Measuring instruments employing immobilized enzyme working electrodes are known for their handiness, rapidity, substrate specificity and other features, and are extending their applications in wide fields including clinical analysis, food analysis and environmental measurements. Among others, the instrument for amperometric measurement, that is, the instrument designed to detect the increase or decrease of electrode active substance caused by the enzyme reaction as the changes in the current output from the working electrode to which a constant voltage is applied, is easy to enhance the sensitivity and is excellent in stability. Accordingly, various electrodes, instruments and methods using the apparatus for amperometric measurement have been developed.
As a representative method of amperometric method, the measuring method by oxygen electrode and the measuring method by hydrogen peroxide electrode are known. These two methods are most widely employed in the immobilized enzyme electrodes, and the hydrogen peroxide electrode method is superior to the oxygen electrode method in response speed and S/N ratio.
In such situation, in the fields of food, fermentation and clinical analysis, there are keen demands for the development of method and apparatus for determination of alcohol, especially ethanol, and various proposals have been disclosed so far. For example, according to the U.S. Pat. No. 4,556,635, it is proposed to use an enzyme electrode obtained by immobilizing alcohol oxidase, in the determination of alcohol content in a water insoluble solvent. However, the activity of alcohol oxidase which is used in the enzyme electrode for alcohol determination and is an enzyme for catalyzing the oxidation of ethanol is relatively low, and its stability is not sufficient practically.
For example, in the initial research examples, by using alcohol oxidase without immobilizing because the activity of alcohol oxidase is low, a method of detecting the catalytic reaction of the enzyme in the solution by an enzyme electrode was proposed (G. G. Guilbault, G. J. Lubrano: Anal. Chim. Acta, 69, 189, 1974). What is interesting in this report is that it is stated that it is hard to detect in the hydrogen peroxide electrode although it is supposed to produce hydrogen peroxide estimating from the reaction mechanism of alcohol oxidase. In this regard, nothing has been reported in detail yet.
Afterwards, the immobilizing method of alcohol oxidase was improved, and the immobilized enzyme electrodes obtained by immobilizing alcohol oxidase have been reported. Such reports include proposal of a method of immobilizing alcohol oxidase by a mild reaction. Inactivation of alcohol oxidase is prevented as far as possible by coexistence of a polymer more likely to react with crosslinking agent, such as glutaraldehyde, rather than with alcohol oxidase, or a method of using a polymer which is made insoluble by pH changes (Japanese Laid-open Patents No. 60-176587, No. 62-215387). These methods are effective to a certain degree to prevent inactivation of alcohol oxidase, but are weak in the bonding force of alcohol oxidase, and involve a possibility of leakage of alcohol oxidase. Hence they are not a sufficient immobilizing method of alcohol oxidase. Besides, these methods are not by nature enough to solve the fundamental problems, that is, the low activity of alcohol oxidase, in particular, the low activity for producing hydrogen peroxide as mentioned above and the lack of stability of immobilized alcohol oxidase.
Yet, these methods could not improve the thermal stability of enzyme electrode obtained by immobilizing alcohol oxidase. The thermal stability is often used as the index of stability of an enzyme, and the enzyme to be used in enzyme electrode should be preferably high in thermal stability as far as possible. If the enzyme is unstable thermally, it is possible to operate the enzyme electrode at low temperature, but at low temperature the enzyme activity is lowered, and it is likely that the sensitivity be lowered. Moreover, control of temperature of the instrument near room temperature is susceptible to fluctuations of room temperature, and it is relatively difficult. Therefore it is desired that the enzyme electrode may operate stably at a temperature about 10.degree. C. or more higher than room temperature. Alcohol oxidase is however, generally inferior in thermal stability, and it is difficult to operate the enzyme electrode using alcohol oxidase stable at such temperature.